Download Polymorphism of the Mitochondrial DNA Control Region in Eight

ISSN 1022-7954, Russian Journal of Genetics, 2008, Vol. 44, No. 7, pp. 793–798. © Pleiades Publishing, Inc., 2008.
Original Russian Text © N.S. Mugue, A.E. Barmintseva, S.M. Rastorguev, V.N. Mugue, V.A. Barmintsev, 2008, published in Genetika, 2008, Vol. 44, No. 7, pp. 913–920.
EXPERIMENTAL
ARTICLES
Polymorphism of the Mitochondrial DNA Control Region
in Eight Sturgeon Species and Development of a System
for DNA-Based Species Identification
N. S. Mugue, A. E. Barmintseva, S. M. Rastorguev, V. N. Mugue, and V. A. Barmintsev
Russian Federal Research Institute of Fisheries and Oceanography, Moscow, 107140 Russia;
e-mail: [email protected]
Received February 6, 2008
Abstract—Intraspecific and interspecific nucleotide sequence variations of the mtDNA control region (D-loop)
were studied with mtDNAs isolated from tissue specimens of more than 1400 sturgeons of nine species: Russian sturgeon Acipenser gueldenstaedtii, Persian sturgeon A. persicus, Siberian sturgeon A. baerii, Amur sturgeon A. schrenkii, Fringebarbel sturgeon A. nudiventris, sterlet A. ruthenus, stellate sturgeon A. stellatus, beluga
Huso huso, and kaluga H. dauricus. The results were used to analyze the interspecific variation of the mtDNA
control region in the given set of species and to develop a test system of ten species-specific primers, which
allowed species identification from noninvasive tissue samples, spawn, and food products of eight species. The
system proved suitable for multiplex PCR. A method was developed for the first time to reliably differentiate
the A. baerii mitotype and the baerii-like mitotype of A. gueldenstaedtii. It was found that, although genetically
separate, A. gueldenstaedtii and A. persicus are relatively young species and have common mitochondrial haplotypes, precluding their identification via mtDNA analysis alone. To develop a system for species identification
of A. gueldenstaedtii and A. persicus, it is necessary to study the polymorphism of nuclear markers.
DOI: 10.1134/S1022795408070065
INTRODUCTION
Sturgeons are a unique ancient group of ganoid cartilaginous fishes and are commercially important. The
family Acipenseridae comprises 27 species, including
two paddlefish species and 25 sturgeon species of the
northern hemisphere. Black caviar and sturgeon products are in great demand and are high-priced, which
leads to excessive fishing and poaching. These factors,
along with destruction of natural breeding grounds as a
result of damming and anthropogenic pollution, have
dramatically reduced the population size of most species in both Eurasia and North America. In 1998, all
sturgeon species were included in Appendix 1 or 2 of
the Convention on International Trade in Endangered
Species of Wild Fauna and Flora (CITES) and, with the
exception of two species, in various categories of the
International Union for Conservation of Nature (IUCN)
Red List [1–3].
Official declaration of the species composition is
mandatory for trans-border transportation of all species
listed in the CITES appendices and products obtained
with such species (including caviar). Since the natural
populations have dramatically decreased in size, sturgeon domestication and artificial rearing become widespread in the majority of black caviar-producing countries, including Russia. Species reared in aquaculture
are often nonnative for the given locality, which complicates species identification. Examples of broad introduction of sturgeons in aquaculture beyond their spe-
cies region are provided by Siberian sturgeon A. baerii
(European Russia, Ukraine, Finland, France, the United
States, China, etc.), American paddlefish (European
countries), and some other species. In addition, rapid
and reliable methods of species identification of surgeon products are essential in view of poaching, illegal
production of black caviar, and falsification of trade
labels and documentation [4].
In spite of the great ecological plasticity and morphological variation, species identification of adult
sturgeons is usually simple and is based on the standard
criteria accepted in ichthyology [5], with the exception
of green (United States) and Sakhalin sturgeons. In
addition, differentiation of Russian, Adriatic, and Persian sturgeons is thought to be problematic. Species
identification of juvenile sturgeons is difficult and
requires special knowledge.
Traditionally, species identification of black caviar
is organoleptic and rather subjective. The caviar taste,
color, and roe grain size substantially vary; the grain
size ranges of different species overlap and are thus
unreliable for species identification. Structural analysis
of the egg envelope has shown that roe grains of different species differ in the shape and structure of micropilus fields, but these traits vary and are hardly distinguishable after roe is treated to produce caviar [6].
Attempts to identify the sturgeon species by biochemical methods have not met with success in general. Isoelectric focusing of roe proteins yields some-
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MUGUE et al.
what different but overlapping patterns for different
species [7, 8]. The difference in albumin spectrum [9]
has not found application.
Molecular methods of species identification of commercial products have been developed for more than
15 years [10]. Genetic identification of sturgeons and
sturgeon products, including caviar, is now one of the
most important proofs acceptable by CITES in importing or exporting the CITES appendix species and relevant products.
The methods of DNA identification can be conventionally divided into two groups addressing nuclear
genetic markers and mitochondrial DNA (mtDNA). An
advantage of the former is that not only pure species,
but also hybrids are identifiable. However, these methods have a drawback of being highly sensitive to the
nuclear DNA integrity and, consequently, are hardly
suitable for analyzing commercial caviar specimens.
The use of mtDNA for analyzing the species composition has several advantages, since mtDNA has a far
greater copy number as compared with nuclear DNA
(especially in eggs) and, owing to its circular structure,
is more resistant to degradation and is better preserved.
Birstein and colleagues [11–13] were the first to
develop a DNA test for species identification of sturgeons. The method took advantage of species-specific
substitutions in a region of the CytB gene and was patented in the United States and Europe as a set of primers allowing species-specific amplification of the CytB
gene fragment. A drawback was that many PCRs were
to be performed and their results analyzed to examine
one specimen. Moreover, almost all primers differed by
a single nucleotide substitution, which allowed false
test results and required that a reference reaction (positive control) be always carried out for each species.
Ludwig et al. [14] further developed the method
based on the CytB polymorphism. The method proposed for species identification involved consecutive
restriction enzyme analyses of a PCR-amplified fragment of the CytB gene with seven enzymes and visualization of the restriction fragments in agarose or polyacrylamide gel.
The above two methods have not been widely
employed, and species identification of caviar and other
products CITES-regulated species are usually solved
by direct sequencing CytB fragments and comparing
the results with the GenBank reference sequences [15].
We were aimed at developing a method to identify
the Russian species of surgeons without sequencing
and restriction enzyme analysis. The main requirements for the method were that it be reliable, simple,
and inexpensive and yield reproducible results in various laboratories with minimum equipment. We studied
the polymorphism of the mtDNA control region (Dloop) in eight sturgeon species in order to identify the
species-specific regions for each species and to design
the primers suitable for PCR-based species identification.
The mtDNA control region is hypervariable as compared with mitochondrial protein- and RNA-coding
genes and, consequently, has not been considered
promising for species identification so far. To isolate its
species-specific sequences, it is necessary to study its
total natural polymorphism and to examine the
sequences of many individuals from various localities
of the species region. Otherwise, primers based on the
individual sequences of one population may yield an
artifactual result for individuals of another populations.
On the other hand, the control region often contains
insertions, deletions, and substitutions of several consecutive nucleotides, which is advantageous for genotyping. Such sites are ideal for designing species-specific primers and developing test systems that are less
sensitive to the PCR conditions and equipment quality.
MATERIALS AND METHODS
To analyze the natural variation of the mtDNA control region, we used sturgeon roe and tissue specimens
(mostly fin fragments) available from the Russian Federal Reference Collection of Genetic Materials. The
collection harbors more than 9000 sturgeon tissue specimens, which were collected in various regions of Russia by researchers of the Department of Molecular
Genetics of Aquatic Organisms, All-Russian Institute
of Fishery and Oceanography or researchers contracted
by the institute. Each specimen is labeled with its species name (as identified by ichthyologists on site), collection site, collection time, and the collector’s name.
The specimens were initially fixed with 96% ethanol in
the field and transferred into fresh ethanol in the laboratory. Every specimen of the collection is accompanied by a detail description of the species, collection
site, population, sex, age, tissue type, and morphometric parameters. The data were included in the collection
protocol, which was signed by the collector. Collection
material is stored in ethanol at –70°ë.
DNA was isolated by standard phenol [16] or salt
[17] extraction after digestion with proteinase K or
maxatase.
The full-length mtDNA control region (D-loop) was
amplified with primers LproF (AACTCTCACCCCTAGCTCCCAAAG) and DL651 (ATCTTAACATCTTCAGTG) directed, respectively, to the tPro
and tPhe tRNA genes flanking the control region.
Amplification was carried out under standard conditions; the annealing temperature was 52°ë. The product
size varied from 950 to 1280 bp, depending on the
82-bp repeat number in the haplotype and the species.
Sequencing was carried out with the same primers,
using a BigDye 1.1 sequencing kit and an ABI 3100
genetic analyzer (United States). Some individuals
were polymorphic for the number of 82-bp repeats at
the control region 3' end, flanked by the proline tRNA
gene. To avoid a superimposition of the sequencing
results obtained with templates differing in repeat num-
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POLYMORPHISM OF THE MITOCHONDRIAL DNA CONTROL REGION
Table 1. Control region sequences examined in this work
Species
Russian sturgeon
Acipenser gueldenstaedtii (typical mitotype)
Russian sturgeon
A. gueldenstaedtii
(“baerii”-like mitotype)
Siberian sturgeon
A. baerii
Fringebarbel sturgeon
A. nudiventris
Stellate sturgeon
A. stellatus
Persian sturgeon
A. persicus
Amur sturgeon
A. schrenkii
Sterlet A. ruthenus
Beluga Huso huso
Kaluga H. dauricus
Number
Number
of sequences of GenBank Total
established
sequences
95
56
151
68
9
77
48
35
83
14
2
16
18
38
56
26
28
54
17
1
18
10
45
18
1
25
2
11
70
20
ber, we used additional primer AHR3 (CATACCATAATGTTTCATCTACC), which was complementary
to the region immediately upstream of the repeat.
In addition to the sequencing data, we used the
sequences available from GenBank (NCBI). The total
numbers of sequences examined and the GenBank
sequences are summarized in Table 1.
We used to SeqMan program to analyze the patterns
for sequence alignment and contig assembly, the
MegAlign program to construct multiple sequence
alignments, and the PrimerSelect program to design the
795
primers; all programs were of the DNAStar software
package (Lasergene).
RESULTS AND DISCUSSION
Variation of the mtDNA Control Region in Sturgeons
We obtained 218 and analyzed 322 nucleotide
sequences (including those available from GenBank) of
the D-loop. Like in most vertebrates, the sturgeon control region had two hypervariable segments (HVS1 and
HVS2) and a relatively conserved spacer between
them. HVS2 harbored 2.5–5.5 repeats of 81–82 bp,
including the TAS motif, which is presumably involved
in initiating mitochondrial heavy-strand synthesis
[18, 19]. Many cases displayed heteroplasmy; i.e., one
individual had several mtDNAs with different repeat
numbers (up to five variants). We studied the mtDNA
inheritance in individuals with several mitotypes differing in repeat number and showed that the unit number
of the 82-bp repeat may change during lifetime and
cannot be used as an informative genetic marker of an
individual.
HVS1 varied in size only in A. schrenkii. A 12-bp
insert was detected in some individuals from the Amur
population. The variation of HVS1 was due to point
mutations and single nucleotide insertions/deletions in
the other species.
Isolation of a conserved region made it possible to
design a primer common for all sturgeon species
(primer AHR, Table 2).
Phylogenetic analysis of the control region
sequences yielded a tree that was similar to earlier dendrograms based on the cytochrome oxidase I gene [20].
The species distinctly clustered into two clades. The
Atlantic clade (of a Pontic–Caspian origin) included
the A. gueldenstaedtii–A. persicus–Adriatic sturgeon
A. naccarii complex, A. baerii, A. nudiventris, A. ruthenus, A. stellatus, and H. huso. The group of Pacific species was also monophyletic and included A. schrenkii
Table 2. Primers employed in species identification of sturgeons
Primer
Sequence
Used with
Product size, bp
AHR
AGF
ABF
TATACACCATTATCTCTATGT
GCACAGACTATGTGGTATCCAGAA
CAGATGCCAGTAACAGGCTGA
AHR
AHR
420
215
ABRM
HusF
DauF
NudF
RutF
SteF
SchF
TGTCTGTCTAGAACATAtG
TATCTATTACCTGCGAGCAGGCTG
CCTCTTATGTACGCGGTGT
TGTCTTTTCTGAAGGAGCTTTGC
GGGAATAACCGTTAATTTGG
GGGGTTCTTGGCATGTTGTGAGCG
TGTGGGGTCACGGAcTTTACAG
ABF
AHR
AHR
AHR
AHR
AHR
AHR
182
374
439
329
190
266
254
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Species specificity
All species
A. gueldenstaedtii
A. gueldenstaedtii
(“baerii”-like)
and A. baerii
A. baerii
H. huso
H. dauricus
A. nudiventris
A. ruthenus
A. stellatus
A. schrenkii
796
MUGUE et al.
1
2
3
4
5
6
7
8
9 10 11 12
1000
500
400
300
200
100
439
420
374
329
266
253
215
190
138
Agarose gel electrophoresis of the PCR fragments obtained for
various sturgeon species. Lanes: 1, marker (100–1000 bp);
2, A. gueldenstaedtii (AGF–AHR, 420 bp); 3, A. gueldenstaedtii with the baerii-like mitotype (ABF–AHR, 215 bp);
4, A. baerii (ABF–AHR, 215 bp and ABF–ABRM, 138 bp);
5, A. ruthenus (RutF–AHR, 190 bp); 6, A. schrenkii (SchF–
AHR, 253 bp); 7, A. stellatus (SteF–AHR, 266 bp); 8, A.
nudiventris (NudF–AHR, 329 bp); 9, H. huso (HusF–AHR,
374 bp); 10, H. dauricus (DauF–AHR, 439 bp); 11, molecular weight marker of the sturgeon PCR products; 12,
marker (100–1000 bp).
and H. dauricus of our sample. Thus, analysis of the
mtDNA control region supports the polyphyletic origin
assumed for the genus Huso (H. huso and H. dauricus)
on the basis of a convergent similarity of morphological
traits in its species, the largest in Acipenseridae.
Construction of a Panel of Species-Specific Primers
Based on the sequences established in this work and
available from GenBank, we identified a strongly consensus sequence with invariable nucleotides for each
species. Multiple sequence alignment of the consensus
sequences allowed us to determine the species-specific
regions of the mtDNA D-loop and to design a set of
species-specific primers. The primers were chosen to
harbor a region that was characteristic of all individuals
of the given species and to lack a region that allowed
dimerization of the primer with itself and primer AHR.
For the convenience of genotyping, the species-specific
primers were at different distances from anchor (common) primer AHR so that the products amplified for
different species would differ in size and would be easily distinguishable in 2% agarose gel (figure). The
panel of diagnostic primers is shown in Table 2.
Discrimination between the A. baerii Haplotype and
the baerii-Like Haplotype of A. gueldenstaedtii
Approximately 30% of A. gueldenstaedtii individuals from the Caspian basin are similar in mtDNA to
A. baerii [13, 21]. The CytB gene sequence does not
allow a discrimination between these two mtDNA
types; i.e., the available molecular methods of sturgeon
species identification fail to reliably distinguish
A. baerii and A. gueldenstaedtii. Since the control
region is four- to fivefold more variable than the CytB
gene, we studied its polymorphism with 56 mtDNA
sequences of A. baerii and 45 baerii-like sequences of
A. gueldenstaedtii. One substitution was found to occur
in the baerii-like haplotype but not in A. baerii.
Taking advantage of this substitution, we constructed primer ABRM (Table 2), which annealed
exclusively to A. baerii mtDNA and, in pair with primer
ABF, allowed amplification of a 182-bp fragment.
Verification of the Test System
The panel of primers was tested with several PCR
kits most commonly used in Moscow (Dialat, Sileks,
Fermentas, etc.), plasticware from several companies,
and several thermal cyclers, including Tertsik MS-2
(DNK-Tekhnologiya) and MJ Research PTC-225
(United States) instruments. The PCR mixture for routine analysis contained 1× Taq buffer (Sileks, Moscow), 2.5 mM MgCl2, 2.5 mM each dNTP (Dialat,
Moscow), 2.5–5.0 pM primers (according to Table 2),
2.8 µl of Cresol–glycerol (3.5 mM Cresol Red, 50%
glycerol in water), 2.5 units of Taq polymerase (Sileks),
2 µl of a DNA extract, and water (milliQ) to the final
volume of 25 µl.
Amplification included initial denaturation at 95°C
for 2 min; 35 cycles of 92°C for 20 s, 57°C for 30 s, and
72°C for 30 s; and last synthesis at 72°C for 10 min.
The sizes of the species-specific PCR products are
given in Table 2. An example of electrophoresis of PCR
products in 2% agarose gel is shown in the figure.
The primers were designed to allow multiplex PCR;
i.e., the reaction mixture can contain more than two
primers. The PCR conditions and the concentrations of
all primers should be optimized in every laboratory,
since they may depend on the instruments, reagents,
and plasticware employed in testing. To identify A.
gueldenstaedtii and A. baerii, we routinely perform
multiplex PCR with primers AGF, ABF, ABRM, and
AHR (1 : 2 : 1.2 : 1) under the same amplification conditions as in single reactions.
The PCR product is applied onto 2% agarose gel
(0.5× TBE) and resolved at 15 V/cm for 30 min. The
species is inferred from synthesis of the amplification
product in the reaction with particular species-specific
primers. PCR with other primers is expected to yield no
product. The products of all reactions carried out with
one specimen can be applied in one well. In this case,
the species is inferred from the size of the PCR product.
For the convenience of species identification, we
obtained a mixture of all possible PCR products (182,
190, 215, 253, 266, 329, 374, 420, and 429 bp). The
mixture is applied on gel together with test samples and
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POLYMORPHISM OF THE MITOCHONDRIAL DNA CONTROL REGION
Table 3. Examination of the test system with specimens
from the Russian Federal Reference Collection of Genetic
Materials
Species
Russian sturgeon
A. gueldenstaedtii
(typical mitotype)
Russian sturgeon
A. gueldenstaedtii
(“baerii”-like mitotype)
Siberian sturgeon
A. baerii
Fringebarbel sturgeon
A. nudiventris
Stellate sturgeon
A. stellatus
Persian sturgeon
A. persicus
Amur sturgeon
A. schrenkii
Sterlet A. ruthenus
Beluga H. huso
Kaluga H. dauricus
Number
Number
of correct
of specimens
identificaexamined
tions
%
480
480
100
152
149
356
356
100
20
20
100
68
68
100
180
180
17
17
100
15
120
25
15
120
25
100
100
100
98*
100**
Notes: * Three specimens were identified as A. baerii (see text for
comments).
** Acipenser persicus was identified as having the typical
A. gueldenstaedtii mitotype, while the sympatric Caspian
A. gueldenstaedtii population displayed the “baerii”-like
mitotype in up to 30% of cases and the typical mitotype
in 70% of cases.
serves as a molecular weight marker, allowing visual
identification of the test product (figure).
The samples and test results are characterized in
Table 3. The method did not yield false results in the
majority of cases, with the exception of discrimination
between the A. baerii mitotype and the baerii-like mitotype of A. gueldenstaedtii (error lower than 1%). The
strong specificity of the diagnostic nucleotide substitution detectable in A. baerii with primer ABRM was verified with large samples of the two species. The test
result was at variance with the declared species in three
cases. The mtDNA control region of the three questionable individuals was sequenced. Two individuals had
the typical A. baerii haplotype and were probably
hybrids (the individuals were collected in a fishery farm
rearing A. gueldenstaedtii, A. baerii, and their hybrids
for a long time). The third case was a reversion: the
mitotype belonged to the cluster of closely related
baerii-like sequences but had the substitution characteristic of A. baerii. Reversions are quite expectable
given the high variation of the mtDNA control region,
and polymorphism for the substitution does not ensure
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species identification with a 100% reliability. With all
false-positive results obtained when testing our system,
the error of identifying A. gueldenstaedtii (both of the
mitotypes) and A. baerii is approximately 1%, which is
allowable is certification of the species composition.
Thus, our test system designed for molecular identification of the eight sturgeon species allows reliable
species identification of sturgeons, caviar, and other
sturgeon products for all but one species (A. persicus).
To identify A. persicus, it is necessary to develop a test
system taking advantage of the polymorphism of
nuclear markers.
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